Process for producing streptomycin



PROCESS FOR PRODUCING STREPTOMYCIN Edwin J. Ratajak, Roslyn, and Robert C. Nubel, Levittown, N. Y., assignors to Chas. Pfizer & Co., Inc., New York, N. Y., a corporation of Delaware No Drawing. Application March 11, 1957 Serial No. 645,023

6 Claims. (Cl. 195-114) This invention relates to a process whereby the production and yield of the antibiotic known as streptomycin may be improved, resulting in a high yield of product at a decreased cost. In particular, it is concerned with the production of streptomycin by fermenting a streptomycin-producing microorganism in a nutrient medium in which the antibiotic may be formed in substantially increased yields by the inclusion of an azoarylsulfonic acid dye selected from the class of azoarylsulfonic acid alkali metal salts.

It is also within the scope of the present invention to provide a culture medium in which this antibiotic may be more effectively produced. It is a further object of the present process to produce a fermentation medium in which an azoarylsulfonic acid salt is introduced which will cause an increase in the amount of streptomycin elaborated. The improvement of the present invention comprises conducting the fermentation in the presence of an azoarylsulfonic acid salt selected from the group consisting of an alkali metal salt of p-(Z-hydroxy-l-naphthylazo) benzenesulfonic acid, an alkali metal salt of diphenyldiazo-bis-a-naphthylaminesulfonic acid and an alkali metal salt of p-dimethylaminophenyl-azobenzenesulfonic acid.

The product called streptomycin is an antibiotic iso lated from the culture of a strain of Streptomyces griseus. It is effective not only against Gram-negative bacteria as Well as Gram-positive bacteria, but also against Mycobacterium tuberculosis. In view of the fact that the free base is unstable, streptomycin is usually prepared in the form of its sulfate or hydrochloride salt. It is to be understood that for the production of streptomycin the present invention is not limited to any particular organism, but it is intended to include the use of variants of any new strain of this species as well as any mutants produced from the described organism by various means, such as x-radiation, ultraviolet radiation, and the. like.

In the past, fermentation media have been used which have given commercially practical yields of streptomycin, but by the improved process herein set forth it is found that the yield of the product is greatly increased and the process rendered much more eflicient. The process described herein for the production of streptomycin by fermenting a nutrient medium with a culture of S. griseus comprises adding to the medium as azoarylsulfonic acid alkali metal salt. In particular, it comprises adding the azoarylsulfonic acid salt to the fermentation medium, inoculating the medium with a culture of S. griseus and permitting growth of this microorganism until a commercially useful quantity of this antibiotic is produced. I

te Patent The salts of the azoarylsulfonic acids which are suitable for the process of this invention are those of the alkali metals such as sodium, potassium, lithium, etc.; the preferred salt of this series is the sodium salt. Only a relatively small amount of such azoarylsulfonic salts need be present, generally in the range of from about 0.20 to 2.5 g. per liter of fermentation medium. It is within the purview of this invention to add the azoarylsulfonic acid salt to the S. griseus fermentation medium, either initially or sometime after fermentation has commenced, thereby obtaining a streptomycin potency which is a substantial increase over that of the control. In general, it is most desirable to add the azoarylsulfonic acid salt to the medium within 24 hours of initiation of the fermentation. j

The fermentation broth must have present in it an organic dye selected from the class of azoarylsulfonic acid alkali metal salts. For this purpose, it is desirable that there be present at least 0.20 g. of dye per liter in the fermentation medium. These azoarylsulfonic acid alkali metal salts are selected from the group consisting of the sodium salt of p-(Z-hydroxy-l-naphthylazo) benzenesulfonic acid (Orange II dye), the sodium salt of diphenyldiazo-bis-a-naphthylaminesulfonic acid (Congo Red dye) and the sodium salt of p-dimethyl-aminophenylazobenzenesulfonic acid (Methyl=0range dye); they are all found to be extremely valuable for use in the process of this invention,

It has also been found that for maximum growth, it is necessary that the pH of the fermentation medium be controlled within a particular range. Highly effective growths may be obtained within the range of from about 5 to about 8. Optimum results are obtained within the range of approximately 6 to 7.5. In addition, the fermion tation process should be conducted under aseptic conditions so as to produce a therapeutically desirable product.

During the fermentation, it is necessary that there be present in the nutrient medium a carbohydrate, such as sucrose, dextrose or starch, or crudenutrients containing these, as a source of energy for the microorganism. It is also necessary that a source of nitrogen, such as ammonium salts, corn steep liquor, casein, soybean meal orpeptones, be present for the sustenance of the microorganism.

The fermentation may take place either on the surface of the quiescent fluid, in shaken flasks, or in deep tanks aided by constant agitation and aeration. For large scale commercial production, the use of deep stainless steel tanks with constant aeration and agitation gives a more rapid and efdcient method of production than is obtained by other means. The cultivation of the microorganism S. griseus preferably takes place at a temperature in the range of 24 to 30 C. A time period of approximately 48 hours for the fermentation has been found most desirable in many instances. In general, a range from about one to five days gives very satisfactory results.

Spores may be used directly as an inoculum, but the most economical production is obtained when preformed inoculum is used in the concentration range between approximately 1 and 5% by volume of the main fermentation medium; A convenient method of operation is to start with an agar slant of the microorganisms and seed shaker flasks containing nutrient media with the Streptomyces griseus from the agar slant.

When Orange II dye is added to the fermentation mixtore, "the session-avers riad;is often increased two fold. In Table I; some typical results are illustrated.

TABLE I Potency of Percent of Amount of Orange 11 Streptomycin Control (v/ml.)

1 355 100 1 924 142 0. 3 252 240 ,1 2 791 20c Orange-II, l: 2 335i 172 2 1 158 S imilarly Congoe R efd serves ftg increase thej amount pf streptomycinproduced. As illustrated in Table IL the f -ollowing typical results are vg'obtained on addition of Congo- Red to the nutrient medium;

" Brea t-I p v Percent'ot Amount 'of-Congo-Re'd 2 Control :No Congo Red (Control);..'.;... 1,500 100 Congo Red, 0.25 g./l 2, 380 158 Congo Red, 0.50 g./1 2,300 180 Congo Red, 0.80 gJl .t 3, 000; 200

The data presented in-these tables show the preferred 30P- erating conditions for ,obtaining. maximum yields with -thegp'articular strainhereiniutilized. r,

Hence, it can be seen from these ;data-that the effect of adding theazoarylsulfonicacidsodium salts to the S. griseus fermentation medium results in-potency increases that are both real and'substantial. p l v This invention is further illustrated byythetollowing examples, which are not to be considered 'as imposing any-limitation thereon.

Example! A nutrient medium, was prepared from the following materials in one liter of tap water:

careless 1 deiitro'se hydrate) Corn deirtrin 5.0 ssa sin e Potassium dihydro n phosphat "0i5 Ziiic' sulfate {nearer 0:044 Magnesiu T 0525 Tapuiater, 1 H 1- 'l tidnfadjusfed toil-1 657 withpotassium hydrox IAfter' the had been- 'adjiistd, 1 0 g; of calcium car- -bonate, 2ml. of'soybean oil antifoam a'ndOzS g. of Dr- "a'nge -II 'dye were addd; and the mixture anto c lavd at atmospheric pressure for about 60. minutes to sterilize the same. After cooling toroom temperature; tlie'nuof Orange II dye per'lit'er of fermentation 4 trient medium in each ferment or "was "then inoculated with with-preformed inoculum described as-above, using 5% by volume, and permitted to grow at 26-27 C. for a maximum time period of about hours with constant agitation and aeration; the latter was accomplished by cultivating the organism aerobically under submerged conditions at an air fiow rate of volume of air per volume of medium per minute. In addition to its other efiect, the agitation maintains the calcium carbonate in suspension. A portion of the final mash was adjusted to a pH of about 3 with sulfuric acid, filtered and assayed. It was found that the amount of streptomycin produced was 3252'y/ml.; this represented a 2.4-fold increase over the control value 1'355 ml.) obtained when the=process is conducted in the ahsenceof anyifira'n-g'e II dye.

*Exaiii p ill The samereeedureas described mammals-I 'was followed except that 0.80 g. of Congo Red dye was present in a" liter "of "the-fermentation mixture. "A streptomycin potency of 3000 y/m1. was obtained; this represented a more than two-fold-increase over'the-control value.

those for the preceding example's to determine the effect of various coricentrationsof OrangeILor Congo Red dyes on the production of stre pitonijyicin;gthe results obtained are presented in 'iahlesl and II. Tl us, 1 g.

i m, a

toms a'streptomycinpotency offZYQI'Y /m of Congo Red dye affords a potency/ of 2:7] /rnh; on

the other hand, 0.25 g. of Oran'g'eII dye per liter aiiords a streptomycin potency of l924'y/ml. w ia foz'slgxoroqiigo ,Red .dye per liter yields a streptomycinpotency of "was obtained over" that of'the control value.

Examplw The "same procedure was followed "as in Example *1 except that the Orange-IIdye was' -added to the'reaction mixture "24 hours afterth'e fermentation had-commenced.

The yield of streptomycin obtained was in the same fange i as that-afforded when the dye was initiallyspres'ent-in the "fermentation medium.

'What'jis claimed is: I 1. In "a-process for "the produ'etion "of-streptomycin "by fermenting a nutrient -medium withastrptomyciri-producing" microorganism, the improvement which comprises conducting the fermentation in'the presence'bfansazo 'ar'ylsulfonic acid saltsel'ectedfrom'the group consisting -of an alkali metal'salt'of p-(2-hydroxy-1=naphthylazo) benzenesulfonic acid, an 'alkali metal salt ofdiplienyldiazo-bis-u-naphthylaminesulfonic acid and an "alkali metal salt of p-dimethyl=amin0phenyla2obenienesulfonic acid.

'2. In 'a-process for the production of streptomycin by fermenting a nutrient medium with aculture of StrepiO'riiyes*Zgriseus, thev improvement which -comprises addring to the'medium within 7 24 hours of v initiation "oftlie fermentation an Iazoarylsulfonic acid salt rselecgted. from "the g'roup consisting of "an 'alkali -metal-salt--of :p 2rhydroxyl n'aphthylazo) b'e'nzenesulfonic acid, yanjallcali metal of *diphenyldiazo-bis-wnaphthylaminesulfonic acid and 'an alkali'metal salt of 'p dimethylaminophenylazob'enzenesulfoni'c acid.

'3. A- process 'as claimed in claim *1 wherein thejazo- 4, A process as claimed in claim 1 wherein the 'azoai'gll- 5 6 sulfonic acid alkali metal salt is the, sodium salt of di- References Cited in the file of this patent phenyldiazo-bis-a-naphthylaminesulfonic acid. UNITED STATES PATENTS 5. A process as claimed in claim 1 wherein the amarylsulfonic acid alkali metal salt is the sodium salt of 2462175 Folkers 1949 p-dimethylaminophenylazobenzenesulfonic acid. 5 255576O Regna June 1951 a 2,555,762 Regna June 5, 1951 6. A process as clauned 1n clalm 1 whereln the az0- 2,560,889 Regna July 17, 1951 arylsulfonic acid salt is present in the concentration range of from about 0.20 to 2.5 g. per liter of the medium 2604472 Regna July 1952 and the process is conducted at a pH in the range of from 10 OTHER REFERENCES about 5 to about 8 at a temperature in the range of 24 Kuehi et al.: Jour. Am. Chem. Soc., 68 (1946), 11 to 30" for a time period of from about one to five days. 1460-1462. 

1. IN A PROCESS FOR THE PRODUCTION OF STREPTOMYCIN BY FERMENTING A NUTRIENT MEDIUM WITH A STREPTOMYCIN-PRODUCING MICROGOORGANISM, THE IMPROVEMENT WHICH COMPRISES CONDUCTING THE FERMENTATION IN THE PRESENCE OF AN AZOARYLSULFONIC ACID SALT SELECTED FROM THE GROUP CONSISTING OF AN ALKALI METAL SALT OF &-(2-HYDROXY-1-NAPHTHYLAZO) BENZENESULFONIC ACID, AN ALKALI METAL SALT OF DIPHENYLDIAZO-BIS-ANAPHTHYLAMINESULFONIC ACID AND AN ALKALI METAL SALT OF P-DIMETHYL-AMINOPHENYLAZOBENZENESULFONIC ACID. 